A Highly Sensitive Assay for Histamine Using Ion‐Pair HPLC Coupled with Postcolumn Fluorescent Derivatization: Its Application to Biological Specimens

Abstract: A simple and highly sensitive method for the determination of histamine (HA) was developed using ion‐pair, reversed‐phase HPLC coupled with postcolumn o‐phthalal‐dehyde derivatization fluorometry, and it was applied to the unpurified extracts of human and rat plasma, and brains of rats and mice. The HA concentrations both in the plasma and brains determined by the present method were well consistent with the values obtained by cation‐exchange HPLC with postcolumn fluorescent derivatization currently in use. The present method was more advantageous than the assay using cation‐exchange HPLC: (1) it was three to four times more sensitive (the detection limit was 0.5 pg of HA), and (2) it enabled the measurement of HA in samples containing C/?)a‐methylhistamine, a potent and specific H3‐receptor agonist, which could not be separated from HA by cation‐exchange chromatography. Using the present method coupled with intracerebral microdialysis, we found in the rat hypo‐thalamus that (R)α‐methylhistamine (5 mg/kg i.p.) markedly decreased the extracellular concentration of HA with a maximal effect (83% reduction) during 30‐60 min after injection, suggesting that most of HA in the microdialysate fraction is neuronal in origin.